Transcriptomic analysis for the retested positive COVID‐19 patients with long‐term persistent SARS‐CoV‐2 but without symptoms in Wuhan

Dear Editor A new type of COVID-19 patients has been reported (referred as LTPPs) recently, who were retested with positive results after hospital discharge, with long-term persistent SARS-CoV-2 in their bodies, although they were recovered from acute infection with no clinical symptom of COVID-19.1,2 They pose new challenges to the prevention and treatment of COVID-19, but the underlying mechanism for such a contradictory phenomenon remains uncovered. To address this issue, we performed transcriptomic analyses on the peripheral blood mononuclear cells (PBMCs) from 12 LTPPs at long-term positive and second recovery stages, with the longest positive time of 132 days (Table S1). They were all diagnosed as moderate patients during the acute infection period (first admission time: January 14, 2020 to March 28, 2020), and then discharged due to two consecutive negative reverse transcription polymerase chain reaction (RT-PCR) results in other hospitals, followed by a 14-day-quarantine-period. However, in the 14-day-quarantine-period, they were recharged to Wuhan Pulmonary Hospital owing to the recurrent positive RTPCR results, although they showed no clinical symptoms of COVID-19 (Table S2). They were final discharged (May 4, 2020 to June 10, 2020) as recovery patients (RPs) according to Chinese discharge standards. Fifteen PBMC samples from healthy-individuals were also included as controls (HCs). Principal component analysis (PCA) showed differential expression ofmRNAs, lncRNAs andmiRNAs (adjusted P-value < .05) among LTPPs, RPs, and HCs (Figure 1A, S1−S2, Tables S3−S5). Further analysis identified 1708, 962 and 1280 differentially expressed genes (DEGs), 1503, 623, and 1218 DElncRNAs, and 437, 341, and 312 DEmiRNAs in the LTPP/HC, RP/HC and RP/LTPP groups, respectively. Here, the majority of DEGs (68.44%) were significantly up-

We then performed Ingenuity Pathway Analysis (IPA) to explore the enriched pathways/biofunctions responsible for LTPPs. The results revealed 77 enriched pathways and 272 enriched biofunctions in LTPPs, most of which (96.10%, 95.22%) were significantly up-regulated ( Figure 1C−F). Among them, the immune-response related enriched pathways (31) and functions (152) ranked first in the LTPP/HC group ( Figure 1E, F), all of which were significantly up-regulated, including many innate-immune related pathways (23) and biofunctions (35), and acquired immune related pathways (14) and biofunctions (11) (Figure 2A,B). This might explain the suppression of the emergence of clinical symptoms of COVID-19 through fighting against the long-term persistent SARS-CoV-2. Significant activation of acquired immune-responses were also indicated by higher proportion of T lymphocyte, normal range of lymphocyte counts, and high neutralization abilities against SARS-CoV-2 ( Figure 2C,D, Figure S3, Table S2).
Why was not SARS-CoV-2 completely eliminated in LTPPs by highly activated immune-responses? We found that all activated immune related pathways/functions in our study have been reported to facilitate the resistance/elimination of foreign pathogens, except "T-Cell Exhaustion Signaling Pathway" (Figure 2A,B). 3 Since Tcell exhaustion can decrease the effector function and proliferative capacity of T-cell, 3,4 it may be a key reason for the long-term persistence of SARS-CoV-2 in LTPPs. Previous studies have also demonstrated that the persistent antigen stimulation from pathogens can result in T-cell exhaustion. 3 This was further supported by the fact that the pathway was recovered to normal in RPs ( Figure 2A). Here, all 22 enriched genes were significantly up-regulated in LTPPs, therefore, the inhibitors for these        HEK293T cells were co-transfected with miRNA mimics/miR-NC (negative control mimic) and 3′UTR-WT (wild type)/MT (mutant) constructs. Empty vector was transfected as a negative control. Firefly luciferase activity was first normalised against Renilla luciferase activity, and relative luciferase activity of miRNA mimic transfected cells was then compared to those of miR-NC transfected cells. The data were presented as the means ± SD (n = 3). Unpaired two-tailed t-test was used. **P < .01, ***P < .001, ****P < .0001.

TA B L E 1
The 38 potential therapeutic targets for the treatment of LTPPs "T-cell exhaustion" related genes (IL6, HLA-G, etc.) provided some promising therapeutic drugs to treat long-term infection of SARS-CoV-2 by reversing the progression of T cell exhaustion, while further studies are warranted. We further determined key hub genes (hereinafter, KHGs) in the transition from LTPP to RP steady-states based on the number of pathways/biofunctions they participated in ( Figure 3A−D), since genes are more important in the contributions for clinical symptoms if they participate in more pathways/functions. 5 We first listed the top-10 KHGs for enriched pathways and biofunctions in the LTPP/RP group, and 21 KHGs were obtained, all of which showed significant upregulation ( Figure 3A−D).
Collectively, through exploring critical pathways and key hub genes in the transition from LTPP to RP steadystates, we screened out 38 potential therapeutic targets (12 cytokines, 6 transcription regulator, 6 transmembrane receptors, etc.) for LTPPs (with shared five genes) in our study, including 22 enriched genes in the "T Cell Exhaustion Signaling Pathway" and 21 KHGs, all of which were significantly activated/up-regulated in LTPPs. Importantly, 26 of 38 genes have been reported to be associated with COVID-19, and six ones (IL6, CSF3, MIF, ATF4, HLA-G and LGALS3) have been documented to be potential therapeutic targets for the treatment of COVID-19 (Table 1), indicating the credibility of our screening strategy. More importantly, 8 of 38 genes showed a continuous trend of down-regulated expression from LTPPs to RPs and then to HCs (IL6, IL10, IL12B, CSF3, IL36G, CCL2, CD274 and CD80), and 14 genes were restored to normal in the RPs (CCL5, LTA, RASD2, WNT5A, IFNG, MIF, ATF4,  GNG5, THBS1, LGALS3, BATF, HLA-DPA1, HLA-DRA and HLA-DRB1), which displayed consistent expression tendency with disease recovery, indicating their potential roles during the transitional process from LTPPs to RPs.
We then identified 65 down-regulated DEmiRNAs targeting the 38 up-regulated targeted genes ( Figure S4, Table  S6), since previous studies indicated that human body could activate/up-regulate some genes to fight against diseases through epigenetic downregulating corresponding miRNAs. 6,7 Importantly, 25 of 65 miRNAs showed reversed expressed trend with corresponding 17 targeted genes ( Figure 3E,F), displaying consistent tendency with disease recovery duration, suggesting their important roles in the recovery of LTPPs. We further focused on seven miRNA-mRNAs pairs with high confidence prediction of targeting relationships, among which four miRNAs (hsa-miR-511-5p, hsa-miR-128-1-5p, hsa-miR-365a-5p and hsa-miR-548o-3p) targeting five mRNAs (RASD2, IL6, BATF, HLA-DRB1 and CD80) were validated using 3′UTRluciferase assay ( Figure 3G, Figure S5). Further functional validations for these genes and miRNAs (especially for unreported ones) and their potential therapeutic values are worthy of further investigation. Some limitations do exist in our study. Validation experiments cannot be performed since we do not have the required qualifications and license for handling SARS-CoV-2. Additionally, due to the serious conditions of COVID-19 pandemic and the huge treatment burden of clinicians in Wuhan during Marchto June 2020, it was very difficult for us to pursue more analyses at that time (e.g., RT-PCR, western-blot, proteomic), which definitely will be helpful in this study. Additionally, the profiles of the COVID-19 patients after vaccination are worthy of further investigation for comprehensively featuring COVID-19.
In summary, our results uncovered the underlying immune mechanism for the contradictory phenomenon of LTPPs: while the significantly activated innate and acquired immune responses in the LTPPs could potentiate the body against long-term persistent SARS-CoV-2 resulting in uncharacterized clinical COVID-19 manifestations, the significantly activated T cell exhaustion pathways might prevent SARS-CoV-2 from complete elimination, leading to their long-term survival in the LTPPs. Our findings also provide the important references and improved understanding of COVID-19.

S U P P O R T I N G I N F O R M AT I O N
Additional supporting information can be found online in the Supporting Information section at the end of this article.